This project will examine the functional consequences of Toll-like receptor-3 (TLR-3) expression as it relates to features of Alzheimer's disease. Toll-like receptors, which are a family of 10 related pattern recognition receptors, have multiple functions in coordinating host defenses to microbial agents. We identified TLR-3 as being prominently expressed by microglia in AD brains, particularly those plaque-associated, while other significant TLR were not associated with glia. As TLR-3 signaling can have different consequences, we consider it timely to investigate in AD and related relevant human model systems. As TLR-3 is primarily an endosomal localized receptor, its function and the consequence of its activation to AD requires investigation. We have developed a central hypothesis based on preliminary results that stimulation of microglia and astrocytes with ligands for TLR-3 will induce anti-inflammatory and protective responses, and also promote A? removal. As stimulation of related endosomal TLR-9 has been suggested as a therapeutic option for removing amyloid from brain, we are positing that TLR-3 activation may have similar outcome. The goal of this project is to identify the nature of microglia and astrocyte responses to TLR-3 ligands in the presence of A?. This will be investigated in vitro in microglia and astrocyte cultures derived from postmortem brains, and in ex-vivo human brain slice cultures. We will also determine if chronic TLR-3 ligand administration to plaque-developing transgenic mice affects A? load and related neuropathology. We will investigate these features in three related aims. Specific Aim 1; We will investigate what are the functional consequences of TLR-3 activation of human microglia and astrocytes on production of proinflammatory and anti-inflammatory cytokines and neurotoxic factors. Cultures from AD and normal brains will be stimulated with a TLR-3 ligand in the presence and absence of A?. We will use antibody arrays to measure cellular responses, and also in vitro neurotoxicity and neuroprotective assays to determine consequences of TLR-3 stimulation. Specific Aim 2; We will investigate what are the functional consequences of TLR-3 activation of human microglia and astrocytes on uptake and degradation of amyloid beta peptide. Both aims will be carried out in vitro using the TLR3- ligand poly IC and/or A? stimulated human microglia and astrocytes described in Aim 1. Specific Aim 3; We will use two complex systems, where interactions of microglia, astrocytes and neurons occur, to investigate the same features as in aims 1 and 2. We will use ex vivo slice cultures of human brains and also amyloid plaque developing transgenic mice to determine whether TLR-3 activation promotes or reduces inflammation and neuronal survival, and increases or decreases glial uptake and degradation of A?.